In recent years, a mass spectrometric method which includes the steps of dissociating an ion derived from a target compound and detecting the thereby generated fragment ions (or product ions) after separating them according to their mass-to-charge ratios has been widely used for the identification or structural analysis of high molecular compounds. For example, an ion-trap time-of-flight mass spectrometer is commonly known as a device for such a mass spectrometry. The most popular technique for dissociating a high molecular ion captured in an ion trap in such a mass spectrometer is a low-energy collision induced dissociation (CID). For the dissociation of the ions derived from proteins or peptides, other various techniques are currently also widely used, such as electron transfer dissociation (ETD) or electron capture dissociation (ECD).
In the case of ETD, negative molecular ions are cast into the ion trap as reactant ions. Within the ion trap, the reactant ion is made to collide and interact with an ion derived from a sample component. Through this interaction, an electron is transferred from the reactant ion to a proton in the ion derived from the sample component, making the proton turn into a hydrogen radical. The radical species of the ion generated by this reaction undergoes a bond-specific dissociation. This reaction can be expressed as follows:[M+nH]n++A−→{[M+nH](n−1)+}*+A→Dissociationwhere M is the target molecule, H is the proton, A− is the reactant ion, and n is a positive integer. The asterisk (*) indicates the radical state.
In the case of ECD, electrons are cast into the ion trap. Within the ion trap, the electron is added to a proton in the ion derived from the sample component. As a result, the proton turns into a hydrogen radical. The radical species of the ion generated by this reaction undergoes a bond-specific dissociation. This reaction can be expressed as follows:[M+nH]n++e−→{[M+nH](n−1)+}*→Dissociation
Unlike collision-type dissociation methods (such as CID), ETD and ECD are radical induced dissociation methods. In this type of dissociation, the fragmentation specifically occurs at the N-Cα bond in the peptide backbone. Therefore, c/z-type fragment ions, which cannot be easily generated by the low-energy CID, are abundantly generated. Additionally, since the fragmentation occurs with the modification sites (such as the sugar chain) retained intact, it is easy to identify the modifier or locate the modification site. This is useful for structural analyses of high molecular compounds.
However, in the ETD or ECD, as is evident from the aforementioned reaction formulae, a singly-charged ion derived from the sample component will be immediately neutralized after the radical reaction. Therefore, theoretically, only the multiply-charged ions having two or more charges can be dissociated. Furthermore, in normal situations, this technique is only effective on positive ions; it is difficult to dissociate negative ions. Additionally, since the valence of the ion decreases with each cycle of dissociation, the operation of repeating the dissociation a plurality of times to generate immonium ions including amino-acid side chains can only be applied to an ion whose valence is equal to or greater than the number of amino acid residues. Still another problem is that the technique requires a charged-particle optical system, such as an einzel lens, in order to cast charged particles (negative ions or electrons) into the ion trap.
The previously mentioned ETD and ECD are radical induced dissociation methods which use charged particles. There is also a radical induced dissociation method that uses a neutral radical particle, which is a kind of non-charged particle, in place of the charged particle. For example, Non Patent Literature 1 and Patent Literature 1 disclose a method in which a sample-component-derived ion being transported in an ambience of atmospheric pressure is irradiated with a hydroxyl radical (OH radical) to dissociate the ion.
However, the aforementioned radical induced dissociation method using the hydroxyl radical is under the constraint that the dissociation is induced in an ambience of atmospheric pressure. In commonly used mass spectrometers, the ion trap is placed in a vacuum atmosphere. Therefore, the aforementioned dissociation method cannot be used for the dissociation of an ion within such an ion trap.
Examples of the radical induced dissociation method using a neutral radical particle in a vacuum atmosphere have also been reported, e.g. in Non Patent Literatures 2 and 3. According to Non Patent Literature 2, an attempt was made to cause a radical induced dissociation in a similar manner to the ECD or ETD by casting hydrogen radicals onto singly-charged peptide ions captured in a cell of a Fourier transform ion cyclotron resonance mass spectrometer (FT-ICR MS), with the conclusion that the dissociation of the ions could not be recognized. Non Patent Literature 3 includes a report that a replication study of the experiment described in Non Patent Literature 2 was conducted, but the dissociation could also not be achieved.
Non Patent Literature 4 and Patent Literature 2 disclose a method in which an ion captured in an ion trap is dissociated by casting an accelerated beam of neutral particles or radical particles into the ion trap by using a fast atom bombardment (FAB) gun. According to the descriptions in those documents, this method works as follows: The particles accelerated to high speeds become excited and emit electrons. Those electrons adhere to the ions captured in the ion trap and induce the dissociation by a similar mechanism to the ETD or ECD.
This dissociation method does not require a charged-particle optical system to cast neutral or radical particles into the ion trap. However, despite the use of non-charged particles, this dissociation method eventually relies on the transfer or capture of electrons to induce the dissociation in a similar manner to the ETD or ECD. Therefore, in principle, the ion to be dissociated must have a charge number equal to or greater than two, which means that singly-charged ions cannot be dissociated.